PROJECT SUMMARY/ABSTRACT While nearly one quarter of the world?s population is latently infected with Mycobacterium tuberculosis, only a small fraction develops active TB. In 2017, 10 million people developed TB, including 1 million children. Risk factors for progression to active TB after aerosolized exposure are, in large part, related to age and immunologic competency. However, improved methods are needed to identify people at highest risk of developing active TB. There is a growing appreciation that the microbiome?comprised of the trillions of organisms that live within the human?plays essential roles in the regulation of host metabolism and immunity. Immune pathways include the ability of commensal organisms to outcompete invasive pathogens and the ability of certain flora to stimulate innate immune responses, subsequently influencing adaptive responses that promote mucosal immunity. While the majority of the microbiome is found in the human intestines, additional important flora reside within various other compartments, most relevant of which includes the lungs; there is an emerging appreciation for the cross-talk between these two compartments, termed the gut-lung axis. Regarding TB, little is understood about ways in which the microbiome may impact risk of progression to active TB or disease outcomes. Although it is well known that the use of antimicrobials leads to a disruption in the intestinal flora, the impacts of first-line TB treatment with rifampin, isoniazid, pyrazinamide and ethambutol on the microbiome are understudied. The recommended course of TB treatment is lengthy?at least six months; therefore, there is a concern that this period of continuous antimicrobial exposure can have long-term effects on the microbiome. In this application, we propose to evaluate the intestinal microbiota from pediatric participants who were recruited in a cohort study of children from rural Tanzania undergoing evaluation for TB disease; we will use stool samples collected at three time points over six months, including a timepoint prior to any TB treatment initiation. We hypothesize that prior to TB treatment initiation, ?cases? with TB will demonstrate perturbations in the intestinal microbiome that are distinguishable from ?control? children without TB. Additionally, serial assessments of the intestinal microbiome will demonstrate how TB treatment is associated with alterations in microbiologic membership and functional potential compared to ?control? children who did not need or receive TB treatment. We will test our hypotheses via the following aims: 1) evaluate the intestinal microbiome among children with TB disease compared children who have been ruled out from having TB disease, and 2) determine the longitudinal impacts of TB treatment on the composition, diversity, and resilience of the intestinal microbiome among children on TB treatment compared to controls. DNA extraction from stool samples and metagenomic sequencing will be conducted at our long-term collaborative research sites in Tanzania, and bioinformatic analyses will be led by the University of Virginia. Successful completion of these aims will highlight ways in which the intestinal microbiome affects TB pathogenesis.